ECOHAB 2004-2005: Project Summaries

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Anderson, D.M. (Woods Hole Oceanographic Institution), C.H. Pilskaln (University of Maine), and B.A. Keafer (Woods Hole Oceanographic Institution). ECOHAB: Alexandrium spp. cyst dynamics in the Gulf of Maine: delivery, deposition, and resuspension. 9/1/04-8/31/07. NOAA Award: NA04NOS4780274. Email: danderson@whoi.edu.

The predominant harmful algal bloom (HAB) problem in the Gulf of Maine is paralytic shellfish poisoning (PSP), caused by the saxitoxin-producing dinoflagellate Alexandrium fundyense. Blooms of A. fundyense are highly seasonal, consistent with the view that life history transformations between cysts and vegetative cells are major regulatory factors. Another cyst-forming, Alexandrium species blooms in the GOM as well -- A. ostenfeldii. This species is of interest because it produces spirolides, a family of highly potent, fast-acting toxins that are more potent than saxitoxins. The ecology and oceanography of HAB species like A. fundyense and A. ostenfeldii have been relatively well studied, but one aspect of their autecolgy remains poorly understood  their encystment and excystment dynamics. Here we propose a study to focus on several aspects of that dynamic  the processes controlling the delivery, deposition and resuspension of the cysts of these two key Alexandrium species in the GOM. The overall objective is to map Alexandrium spp. cysts over a broad-scale in the bottom sediments and benthic nepheloid layer (BNL) of the GOM, and to relate these distributions to dynamic processes such as resuspension, germination, and encystment fluxes. Specific tasks are to: 1) Develop molecular tools for identification and enumeration of A. fundyense and A. ostenfeldii cysts; 2) Update and expand an existing regional Alexandrium fundyense cyst map to include key cyst accumulation zones in the GOM, as well as the toxigenic A. ostenfeldii; 3) Map the thickness and Alexandrium cyst concentration of the BNL across the study area; 4) Obtain time series data on resuspended cyst abundance within the BNL at two key locations in the GOM; 5) Quantify the time-varying flux of Alexandrium cysts in the upper water column and the time-varying cyst resuspension flux near two cyst deposition zones in the GOM and relate these fluxes to water column samples of Alexandrium vegetative cell abundance and the observed abundance of cysts in bottom sediments; and 6) conduct model runs to assess the relative importance of cyst germination from bottom sediments and the BNL and to assess the role of cells germinating from deep, offshore basins in the bloom dynamics of Alexandrium spp. in the GOM.

Significance of the proposed study to the overall ECOHAB Program goals: The project will address significant questions related to understanding the sources of HABs and the physical processes that influence their transport and fate. Specific focus on resuspension of cysts as a mechanism by which cysts may be transported from bottom sediments into the overlying water column, or by which newly formed cysts may be maintained in near-bottom waters, will enhance predictive capability of models leading to HAB forecasting. Project results and their incorporation into regional physical modeling efforts will produce an up-to-date and expanded cyst map for the GOM that will include information on regions of resuspended cyst reservoirs.

 


 

Caron, D.A. (University of Southern California, Los Angeles) and P.E. Miller (University of California, Santa Cruz). ECOHAB: Stimulation of toxic blooms of the diatom Pseudo-nitzschia spp. by urban river discharge into southern California coastal waters. 1/1/05-12/31/07. EPA Award: RD831705. Email: dcaron@usc.edu.

This is a field-oriented research program to investigate the relationship between freshwater inputs from a highly urbanized region (southern California from the Palos Verdes peninsula to Long Beach) to the growth of Pseudo-nitzschia spp. and the production of domoic acid by members of this diatom genus in the adjacent coastal ocean.

Objectives/Hypotheses: Freshwater discharge into the Southern California Bight is strongly episodic, highly channelized, and restricted primarily to the winter/early spring. These freshwater inputs contribute substantial amounts of inorganic nutrients, labile organic compounds and trace metals to coastal ecosystems. It is hypothesized that these meteorological events greatly influence phytoplankton dynamics and the formation of harmful algal blooms in these waters. This research program will examine the connection between storm events and Pseudo-nitzschia species success and the production of domoic acid in coastal communities.

Approach: A sampling grid (up to 45 sites) encompassing three major river discharges will be studied following a major rainfall event in each of two consecutive years. Samples will be collected at 2-4 day intervals for a period of 2-3 weeks following each event. Supplemental samples will be collected bi-weekly throughout the year along a single cross-shelf transect. Remote sensing will be used to guide sampling during the storm events. Plankton abundances will be determined using flow cytometry, FlowCAM and fluorescence microscopy. Pseudo-nitzschia spp. abundance will be determined by microscopy (light, SEM), fluorescent in-situ hybridization, and quantitative real-time PCR. Domoic acid concentrations will be obtained using an immunological method. Physical parameters, nutrient and trace metals will be analyzed. Expected

Results: This project will discern the patterns of environmental and biological factors stimulating population growth and domoic acid production by Pseudo-nitzschia species in coastal waters of southern California. Key factors leading to blooms of these species and toxicity events will be documented.

 


 

Coats, D.W., M.R. Sengco (Smithsonian Environmental Research Center) and D.M. Anderson (Woods Hole Oceanographic Institution). ECOHAB: Role of parasitism on HAB dynamics: Amoebophrya sp. ex Alexandrium tamarense. 8/1/05-7/31/08. NOAA Award: NA05NOS4781193. Email: coats@serc.si.edu

Harmful algal blooms (HABs) develop from the rapid growth and accumulation of certain microalgal species, and cause many deleterious impacts on human health, aquatic organisms, important industries, and the quality of freshwater reservoirs and coastal environments. Globally, HABs have seen a dramatic increase in frequency, magnitude, distribution and impacts in recent years, which has prompted considerable interest in processes that regulate their formation, persistence, and decline. Trophic interactions, like parasitism, may play a significant role in bloom dynamics. Species of Amoebophrya are particularly noteworthy, as they are widely distributed in coastal environments, with infections known for numerous host taxa. Infections prevent reproduction of the host, are short in duration, and invariably result in death of the host, all of which make these parasites likely candidates for controlling host populations. These organisms are even viewed as potentially useful agents for the biological control of HABs.

This proposal focuses on a recently-isolated dinoflagellate parasite from the genus Amoebophrya and its interaction with its host, Alexandrium tamarense, a bloom-forming, toxic dinoflagellate that impacts U.S. coastal waters. Our first objective is to characterize the parasite in culture to fully establish its identity and relationship to the genus Amoebophrya. This work includes basic parasite morphology/cytology throughout its entire life cycle, and molecular phylogenetic analysis. In conjunction with phylogenetic analysis, our second objective is to develop a series of molecular probes, which are Amoebophrya-specific and specific to our isolate, for later use in field studies. Our third objective is to study host-parasite interactions in the laboratory. This work examines parasite generation time under various conditions: host-parasite ratio, growth phase of host, dinospore age, and environmental conditions (i.e. light, temperature, salinity). The data from this work will be used in modeling the dynamics of host and parasite populations. Our fourth objective is to examine the host range and preference of our isolate. Our fifth objective is to determine the fate of saxitoxins from A. tamarense during and after infection. Lastly, our sixth objective is to study the dynamics of the parasite and A. tamarense (and other dinoflagellate hosts) in the field over time and space to determine the role of parasitism on host populations.

Overall, this research seeks to further our fundamental understanding of this little-known group of parasitic organisms, and to investigate the role these parasites play in affecting host population dynamics in the laboratory and field. These studies, together with practical and ecological considerations, may allow us to evaluate the strategy of using this parasite as a biological agent to control HABs like those produced by A. tamarense.

 


 

Dam, H.G., G. McManus and P. Kremer (University of Connecticut). ECOHAB: Linking food web structure, grazer toxin resistance and ecological stoichiometry in understanding harmful algal blooms. 1/1/05-12/31/07. EPA Award: RD831706. Email: hans.dam@uconn.edu.

Objectives/Hypotheses: The complex dynamics and feedbacks of planktonic food webs determine the formation and fate of harmful algal blooms (HAB), and the trophic transfer of toxins. In principle, both bottom-up forcing (nutrient availability), which constrains the upper limit of plant productivity, and top-down forcing (grazing pressure), which keeps this productivity from reaching its maximum, control HAB. In the simplest case, depletion of top predators and enhanced nutrient supply due to eutrophication can account for the increase of plant production (including HAB) in coastal regions. However such prediction is biased if it ignores three feedback factors seldom considered in tandem in HAB studies: (1) the toxicity of the algae; 2) toxin resistance of grazer populations; and 3) the elemental stoichiometric (C: N: P) imbalance between algae and grazers. Three hypotheses involving these feedbacks will be tested. The first two hypotheses apply to conditions in which the algae are nutrient rich. H1: Trophic cascades are stronger in the presence of toxic algae. H2: trophic cascades are weaker in the presence of toxin-resistant grazer populations. H3: The strength of trophic cascades depends on the interaction of the stoichiometric imbalance of the grazers, the toxicity of the algae and the complexity of the food web.

Approach: These three hypotheses will be tested in combination of controlled laboratory and mesocosm experiments. Rigorous experimental tests for toxic effect of prey on grazers will be run. Comparative and manipulative trophic cascade studies will also be run with simple food webs consisting of several trophic levels with mixtures of toxic and nontoxic foods, under nutrient replete and depleted conditions, and facing toxin resistant and nonresistant metazoan grazer populations.

Expected Results: There is an immediate and urgent societal need to understand what factors govern HABs in order to develop effective HAB mitigation strategies. This work will provide some of the required tools to predict under what conditions HAB happen and to what extent the strength of trophic cascades involving toxic algae are modified by toxin-resistant grazers and the elemental composition of grazers and algae. This knowledge is essential for properly designing adequate mitigation plans for toxic algal blooms.

 


 

Frost, B.W., R.A. Horner (University of Washington, Seattle), C.L. Greengrove, J.E. Gawel, and K. Sian Davies-Vollum (University of Washington, Tacoma). ECOHAB: The relationship between paralytic shellfish toxins and Alexandrium cysts in Puget Sound, Washington. 9/1/04-8/31/07. NOAA Award: NA04NOS4780273. Email: frost@ocean.washington.edu.

This study addresses the relationship between paralytic shellfish toxins in shellfish based on historical records from the Washington Department of Health and the distribution of cysts and vegetative cells of the dinoflagellates Alexandrium spp. in Puget Sound, Washington. Our hypotheses are 1) cysts of Alexandrium spp. occur throughout Puget Sound; 2) cysts are viable and able to germinate (excyst); 3) cysts are resuspended at constrictions between basins; and 4) PSP is produced by motile cells from germinated cysts. A combination of field surveys and laboratory experiments will be used to test our hypotheses. Surface sediments at 31 sites distributed throughout Puget Sound will be surveyed for cysts. Cysts will be identified, counted, isolated, germinated in culture, and the resulting motile cells tested for PSP production. Water samples collected at the same time will be analyzed for the presence of cysts and motile cells of Alexandrium. Sites where cyst concentrations are high will be reexamined for sediment accumulation and mixing parameters. Sites at constrictions into bays and at sills will be sampled to determine where cyst accumulation, turbulence, and resuspension occur. Longer cores will be examined to determine the chronology of cyst appearance and its relationship to the spread of PSP. Sediments will be analyzed for grain size, metals that might influence the germination of cysts, and 210Pb levels to determine possible age, bioturbation and remixing of surface sediments. Cyst occurrence and viability will be related to physical (temperature, salinity, sediment type) and chemical (oxygen, nutrients, sediment metals) factors that might affect their germination and thus bloom initiation. Sites of seedbeds for potential bloom initiation that need additional monitoring for toxins will be identified, areas of cyst resuspension will be determined, and possible means by which Alexandrium has been spread throughout Puget Sound will be suggested.

 


 

Hoagland, P., D. Jin, H.L. Kite-Powell, A. Solow (Woods Hole Oceanographic Institution), G. Herrera (Bowdoin College) and B. Keafer (Woods Hole Oceanographic Institution). ECOHAB: Economic impacts of HAB events and the value of scientific predictions. 9/1/04  8/31/06. NOAA Award: NA04NOS4780270. Email: phoagland@whoi.edu.

The main goal of our proposed research project is to develop a more complete understanding of the ways in which commercial shellfishermen, downstream processors and customers, and government resource managers respond to HAB events in the Gulf of Maine. This understanding will allow us to develop a framework for estimating economic impacts from specific HAB events. A subsidiary but closely related goal is to use this understanding to demonstrate the value of scientific predictions of HAB events. In particular, a thorough understanding of the types, size, and incidence of economic damages will help to clarify the value of public investments in a predictive capacity.

 


 

Kraeuter, J.N. (Rutgers University), V.M. Bricelj (National Research Council, Canada), E. N. Powell (Rutgers University), E.E. Hofmann, J.M. Klinck (Old Dominion University), J. E. Ward (University of Connecticut), and M.D. Gastrich (New Jersey Department of Environmental Protection). ECOHAB: Effects of Aureococcus anophagefferens brown tides in coastal lagoonal systems: Coupling numerical simulation modeling with field and laboratory studies to determine population effects on the hard clam, Mercenaria mercenaria. 9/1/04-8/31/07. NOAA Award: NA04NOS4780275. Email: kraeuter@hsrl.rutgers.edu.

Blooms of the brown tide alga Aureococcus anophagefferens have become common in shallow coastal estuaries in the mid-Atlantic US. The Barnegat Bay/Little Egg Harbor system, part of the EPA Estuary Program, has experienced brown tide blooms in 5 of the last 8 years. These blooms inhibit feeding of suspension-feeders and have caused recruitment failure and mass mortalities of commercially important bivalves, including mussels and bay scallops in the region. The hard clam, Mercenaria mercenaria, an ecologically and commercially important species, is documented as sensitive brown tide. Population level effects of short-term bloom events on a long-lived species such M. mercenaria, are difficult to evaluate experimentally. Cumulative sublethal or lethal effects, especially on early life history stages, may not be evident at the population level for many years. In complex systems such as estuaries, it is difficult to isolate population levels effects due to environmental fluctuations and fishing mortality from other factors. Numerical simulation models offer a means to evaluate the relative significance and cumulative effects of these multiple factors. Such a model has been constructed for the hard clam (Hofmann et al., 2003), and preliminary work has incorporated the effects of brown tide, as far as they are known. Initial simulations show that model predictions are sensitive to brown tide effects on early life history stages, for which there is limited information. This additional information could substantially improve the reliability of model predictions.

This project will combine experimental and modeling efforts to explicitly examine the effects of brown tide on hard clam population dynamics in the Barnegat Bay system. We will experimentally determine effects of brown tide on survival, growth and metamorphic success of hard clam larvae. In addition we will evaluate the size-specific effects of varying concentrations and duration of exposure to Aureococcus on growth and survivorship of hard clam juveniles in the field and in the laboratory. Lastly we will compare the toxicity of various Aureococcus isolates and examine the effects of various concentrations of brown tide in mixed suspensions on feeding activity of adult hard clams using video-endoscopy.

The results of these experimental manipulations will be used to update and refine the existing hard clam model with a larval submodel larval component that simulates the brown tide effects on rates and processes affecting clam recruitment. The model will then be utilized to evaluate the timing and severity of blooms on the individual, year class and population levels relative to other environmental factor such as seasonal temperature variation, changes in food availability, climate change, and fishing pressure in a typical east coast lagoonal estuary.

 


 

  • Larkin, S.L. and C.M. Adams (University of Florida). ECOHAB: Economic effects of HABs on coastal communities and shellfish culture in Florida. 11/1/04-10/31/06. EPA Award: RD83-1707. Email: slarakin@ufl.edu.

    Objectives: (1) To estimate the change in gross revenues to various business sectors of coastal communities affected by HAB (e.g., red tide) events (e.g., test whether changes in restaurant sales are statistically different during periods of red tide and whether the changes are community specific or vary over time);
    (2) To calculate the costs incurred by coastal communities to address the effects of HAB events including planning efforts, contingency plans, beach patrols and cleanup, etc. (which will allow for a test of a minimum community expenditure level); and
    (3) To quantify the effects of HABs and HAB-related harvest regulations on commercial molluscan shellfish operations (which will allow for an evaluation of proposals to alter water quality standards for shellfish harvesting areas). Empirical application will be restricted to Florida for manageability and reduced costs.

    Experimental Approach: Study will use a combination of primary and secondary data, analyzed with econometric techniques and statistical measures. Objective 1 will involve the identification of business sectors impacted by HAB events, such as beachfront lodging and restaurants. A time series of taxable sales will be combined with data on weather (precipitation levels, major storm events, etc.) and HABs (presence and intensity). Municipal and county-level managers and molluscan shellfish (hard clam) culturists will be surveyed following small focus group sessions to identify all HAB-related activities and effects. The managers will be interviewed by telephone to solicit specific information on costs associated with HABs. Culturists will be surveyed by mail to obtain specific information on shellfish losses, harvest closures, cash flow disruptions, etc.). The resulting information will be used to compile a matrix of HAB costs and impacts incurred by coastal communities in Florida.

    Expected Results: The methodologies developed will have broad applicability for investigating the economic effects of HABs. The findings will allow coastal resource managers, local businesses and HAB researchers to better assess the community and business costs resulting from HAB events. This information will provide for a more accurate evaluation of the costs and benefits associated with HAB mitigation, monitoring, regulation, and clean-up efforts. It will also provide data for subsequent analysis of economic impacts.

     


     

    Lin, S. and H. Zhang (University of Connecticut, Groton). ECOHAB: Development of PCR and immunofluorescence methods to measure HAB cell concentration and cell division rate: Karlodinium micrum as a model system. 8/1/05-7/31/08. NOAA Award: NA05NOS4781196. Email: senjie.lin@uconn.edu.

    To monitor dynamics of harmful algal blooms (HABs) and to study potential environmental factors regulating the blooms, it is crucial to simultaneously estimate cell concentration and in situ growth rate (i.e. cell division rate, CDR) at the early stage of bloom development. Although molecular techniques are increasingly used in HAB studies, application of such a powerful technology is still limited to a small number of species, especially in the case of estimation of CDR. A Targeted Individual Study is proposed here to develop a PCR and immunofluorescence (IF) detection system that will allow simultaneous quantification of cell concentration and in situ CDR for Karlodinium micrum. First, extending from the PIs previous research, a dual-gene (mitochondrial cytochrome b-ribosomal RNA) PCR primer set will be developed for quantifying cell concentration (and preliminary survey will be done using existing DNA samples from various estuarine systems). Second, using the proliferating cell nuclear antigen gene (pcna) recently cloned in the PIs laboratory, antibodies will be produced to allow immunofluorecence and estimation of CDR. Meanwhile, empirical correlation between pcna gene expression and growth rate will be attempted as a second way of CDR estimation. Utility of these probes will be assessed using laboratory cultures and field-collected samples spiked with cultured K. micrum. For field application, water samples will be preserved separately for DNA extraction and immunofluorescnece. DNA will be extracted and used in the dual-gene PCR to measure cell concentration. Immunofluorescence of PCNA will be performed and results be applied to a previously established equation for estimation of CDR. In parallel, RNA will be extracted and pcna expression measured using Real-Time quantitative RT-PCR, and CDR will then be estimated using the empirically derived correlation equations. The estimated CDR will be compared with actual CDR measured from the cultures based on cell counts to evaluate the accuracy of the PCR and IF methods. Results from this study will provide specific PCR primers and antibodies and protocols for studying population dynamics and CDR for K. micrum. Results from this study will also provide a framework based on which similar monitoring protocol can be established for other HAB species. In addition, the novel information achieved from this study will be incorporated to courses taught by the PI.

     


     

    Hans W. Paerl, H.W. (University of North Carolina, Chapel Hill), V.J. Paul (Smithsonian Marine Station, Fort Pierce), J.W. Burns (PBS&J), and J.M. ONeil (University of Maryland Center for Environmental Science). ECOHAB: LyngbyaHAB: Ecology of toxic marine cyanobacteria Lyngbya spp. in Florida estuarine and coastal waters. 8/1/05-7/31/08. NOAA Award: NA05NOS4781194. Email: hans_paerl@unc.edu.

    Benthic cyanobacterial (blue-green algal) HABs are becoming more numerous, widespread and persistent in tropical, subtropical and temperate marine embayments, estuaries, and reef environments. Blooms can have many negative impacts, such as directly overgrowing and smothering seagrass, shellfish habitats, and coral reefs. Some nuisance taxa produce toxins and other bioactive metabolites. A genus of particular concern is the filamentous, non-heterocystous nitrogen-(N2) fixing cyanobacteria Lyngbya, species of which (e.g. L. majuscula) are distributed worldwide, especially in the tropical and subtropical oceans. In U.S. coastal waters, Lyngbya blooms have been responsible for fouling large segments of Florida estuaries and bays, the most obvious being Tampa Bay, near-shore reef environments of the Florida Keys, and reefs off of the southeastern coast near Broward County. There are increasing numbers of reports on finfish and shellfish disease and kills as well as human maladies (skin irritations, intoxication) associated with outbreaks of Lyngbya majuscula blooms and other cyanobacterial bloom species. Coastal cyanobacterial blooms are thought to be associated with eutrophication and hydrologic modification of freshwater discharge to marine environments. The following objectives will be carried out in an interdisciplinary effort focused on Tampa Bay, FL (Shell Key): 1) Nutrient additions in in situ bioassays to determine the nutrient(s) that stimulate Lyngbya and other cyanobacterial blooms; 2) Characterize and isolate toxins and other bioactive secondary metabolites produced during bloom and non-bloom events; 3) Develop standards for lyngbyatoxins and aplysiatoxins, 4) Sequence the N2 fixing gene nifH, to examine potential relationships between specific Lyngbya strains, their N2 fixing activity, toxicity, growth and proliferation within Tampa Bay and other Florida Lyngbya populations.

    Parameters we will investigate have been linked to human perturbations of estuarine nearshore environments; hence research products can be evaluated and applied in the context of developing future nutrient and hydrologic management strategies for these habitats and well as risk management plans for humans exposed to Lyngbya toxins. Research results are applicable to subtropical and tropical ecosystems nationally (Hawaii, American Samoa, Guam, Puerto Rico, U.S. Virgin Islands, Gulf of Mexico and S.E. Atlantic) and internationally (e.g., Australia, South Pacific, Caribbean).

     


    Parrow, M.W. and J.M. Burkholder (North Carolina State University). ECOHAB: Cell reproduction, the diel DNA cycle, and sexuality in Karlodinium micrum: life cycle processes fundamental to the ecology of a toxigenic estuarine dinoflagellate. 9/1/04-8/31/06. NOAA Award: NA04NOS4780272. Email: mwparrow@unity.ncsu.edu.

     

  • Karlodinium micrum is a toxigenic dinoflagellate that has formed blooms associated with fish kills in temperate estuarine waters and aquaculture facilities in North America, Europe, and Africa. Although this widespread harmful alga has been studied for many years, information on the asexual proliferation and cell cycle of K. micrum is limited, and its life history is virtually unknown: it is unknown whether sexual reproduction occurs, or if sexuality leads to the formation of dormant cysts as a seed population for future blooms, as occurs in many photosynthetic dinoflagellates. Knowledge of these cell cycle and life history features is fundamental to understanding the basic occurrence, ecology, and bloom formation of K. micrum. The proposed research addresses two overall objectives: First, we will examine cell reproduction in multiple strains of K. micrum, and the diel DNA cycle patterns and the intrinsic growth rate of the species from measurements of population-level DNA synthesis. We will test the hypothesis that cell growth, DNA synthesis, and mitosis in K. micrum follow a typical eukaryotic cell cycle pattern (G1, S, G2+M) that can be related to growth conditions and nutritional cell cycle controls. Second, we will examine the sexual life cycle of K. micrum, and the influence of environmental factors on sexuality. We will test the hypotheses that the sexual cycle of K. micrum influences the population dynamics and ecology of the species, and that environmental factors influence sexuality. Cultures of four K. micrum strains will be examined for cell reproduction and sexuality by light and scanning electron microscopy, and for cell DNA content and population DNA distribution by flow cytometry. Cultures in logarithmic and stationary growth will be profiled over time to measure the rates and patterns of DNA cell cycle progression and restrictions, and intrinsic growth rates for the species will be calculated from cell cycle data. The sexual life cycle of K. micrum will be examined with supporting cell DNA measurements, and the expression of sexuality will be studied in relation to population growth phase and different nutrient (N, P), temperature, and light regimes. This research will advance understanding of the basic cell and life cycle processes that fundamentally influence the ecology of K. micrum. It directly addresses primary needs identified by the ECOHAB program to characterize harmful algal species, reproduction, life stages, and environmental controls on population distribution and abundance.

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    Place, A.R., J. Adolf, and T. Bachvaroff (University of Maryland Biotechnology Institute). ECOHAB: Consequences and causes of variable toxicity in Karlodinium micrum  a cosmopolitan ichthyotoxic dinoflagellate. 9/1/04-8/31/07. NOAA Award: NA04NOS4780276. Email: place@umbi.umd.edu.

    For decades, high densities of the dinoflagellate Karlodinium micrum have been associated with aquatic faunal mortalities worldwide. Recently we have described several toxic compounds (karlotoxins, KmTx) from K. micrum, both in the laboratory and in the field, with hemolytic, ichthyotoxic, and cytotoxic properties which may explain some of the observations associated with high densities of this organism. Our research has revealed substantial variability in toxin yields for different isolates (ca. 0.1 - 1 pg cell-1). Moreover, samples collected during fish kills have contained 10-100 fold this amount on a per cell equivalent (10-12 pg cell-1). We find that a geographic strain variation exists in the toxin produced among K. micrum populations from Southeastern estuaries of the United States. All K. micrum isolates and samples from the Chesapeake Bay contained KmTx1 while all strains from North Carolina to Florida contained KmTx2. Cellular toxicity occurs through non-selective permeabilization of plasma membranes, leading to osmotic cell lysis. Susceptibility to karlotoxins is determined by membrane sterol composition, which also appears to underlie K. micrum's immunity from its own toxins. It is our fundamental premise that K. micrum populations have an extensive variability in toxin production, both in terms of amount and type, and that karlotoxins are primarily produced to aid in prey capture. The proposed work will pose the following questions:

    • Do environmental conditions and/or genotype modulate toxin production? (Project #1)
    • Does genetic strain variation exist within and between populations of K. micrum, and does this genetic variation vary during bloom events? (Project #2)
    • Do differences in the structure of KmTx1 and KmTx2 correlate with biological activity (Project #3)

     


    Sarnelle, O., S. Hamilton, J. Rose, S. Peacor (Michigan State University), and H. Vanderploeg (NOAA Great Lakes Environmental Research Laboratory). ECOHAB: Complex interactions between harmful phytoplankton and grazers: variation in zebra mussel effects across nutrient gradients. 1/1/05-12/31/07. EPA Award: RD831708 and NOAA support to GLERL. Email: sarnelle@msu.edu.

    Filamentous and colonial cyanobacteria are the most important taxa causing harmful phytoplankton blooms in freshwater. A long-standing tenet in lake ecology is that summer blooms of harmful cyanobacteria are a characteristic response to nutrient enrichment and a symptom of eutrophication. However, recent events in the Great Lakes region suggest that the invasion of the zebra mussel (Dreissena polymorpha) is altering well-established functional relationships between nutrient loading and cyanobacterial dominance via the promotion of Microcystis aeruginosa, a toxic species of cyanobacteria, in low-nutrient lakes. We examine the interaction between D. polymorpha and M. aeruginosa with large-scale field manipulations of mussel density and nutrients coupled to research aimed at elucidating underlying mechanisms.
    Objectives/Hypotheses:
    1) experimentally determine whether the effect of D. polymorpha on M. aeruginosa changes direction across a broad gradient of phosphorus loading;
    2) identify thresholds in P loading at which the D. polymorpha effect changes direction;
    3) understand the mechanisms underlying the complex interaction between D. polymorpha and M. aeruginosa, with the explicit goal of predicting the consequences of changes in nutrient loading on harmful phytoplankton abundance in invaded habitats;
    4) determine the degree to which experimental results from inland lakes are relevant to the interaction between D. polymorpha and M. aeruginosa in the western basin of Lake Erie; and
    5) determine the extent to which D. polymorpha promotion of M. aeruginosa translates into increased levels of cyanobacterial toxin levels in the Great Lakes.
    Approach: The centerpiece of the project is a set of three enclosure/mesocosm field manipulations that test the interactive effects of phosphorus availability and Dreissena on M. aeruginosa biomass and dominance. These factorial experiments will be conducted in Gull Lake and Lake Erie. The mechanistic component will include the development of a new theory of herbivore-phytoplankton interactions that can explain negative, positive and neutral effects of an herbivore on the abundance of a harmful phytoplankton species, quantification of selective grazing and per capita nutrient excretion by zebra mussels under widely varying environmental conditions, genetic characterizations of M. aeruginosa via HIP-PCR, and monitoring of cyanobacterial toxin production.
    Expected Results: We will determine whether the effect of D. polymorpha on M. aeruginosa changes direction across a broad gradient of phosphorus loading, and if so seek to identify critical loading thresholds that can be applied in the management of invaded habitats. We expect to achieve a better general understanding of the mechanisms underlying herbivore-nutrient-phytoplankton interactions. Measurements of toxin levels will directly quantify a potentially critical threat to public health, and when coupled to genetic characterizations of Microcystis, should further our ability to predict when and where toxic blooms are likely to occur in Dreissena-infested habitats. Our research may begin to shed light on the question of why Microcystis aeruginosa appears to be uniquely responsive to Dreissena invasion relative to other phytoplankton species.

     


     

    Schultz, I.R., D. Woodruff, and A.D. Skillman (Battelle Pacific Northwest Division). ECOHAB: Domoic acid kinetics and trophic transfer in shellfish: an integrated laboratory and estuarine mesocosm study. 1/1/05-12/31/07. EPA Award: RD31703. Email: irv.schultz@pnl.gov.

    We hypothesize that physiologically based pharmacokinetic models mathematically analogous to the type developed in vertebrates can be adapted for marine invertebrates based on the known physiology of decapod crustaceans and bivalve mollusks. These models will be used to predict the uptake and disposition of the marine algal toxin domoic acid in Dungeness crabs (Cancer magister), Pacific razor clams (Siliqua patula) and blue mussels (Mytilus edulis). Validation of individual kinetic model predictions and trophic transfer of domoic acid will be achieved through a combination of focused laboratory experiments and the use of large-scale estuarine mesocosms containing razor clams and Dungeness crabs.
    Approach: We have successfully applied recent improvements in analytical detection of domoic acid to study the excretion of the toxin in shellfish after intravascular injection and repetitive hemolymph removal. This technique will be used in conjunction with controlled laboratory feeding studies to develop a detailed data set on the uptake, tissue distribution and elimination of domoic acid in shellfish. Physiologically based kinetic models will be developed and specifically parameterized for crabs and bivalves using a combination of recently published and experimentally determined values for the cardiovascular and gastrointestinal / digestive systems of shellfish. Validation of model predictions will initially be performed from indoor laboratory studies and then from the results of a large scale estuarine mesocosm containing razor clams and crabs. In the mesocosm study, 500 clams verified to contain domoic acid will be collected from contaminated Washington State coastal sites. The clams will be added to the mesocosm along with adult Dungeness crabs (previously unexposed to domoic acid), which will feed on the clams. Individual clams and crabs will be repetitively monitored for hemolymph concentrations during the study. Crabs will also be intravascularly injected with 15N-labeled domoic acid to allow simultaneous determination of elimination and uptake of domoic acid. Expected Results: The validated models of domoic acid kinetics in shellfish will provide researchers and risk assessors a useful tool for exploration of mechanisms controlling selective retention of domoic acid by certain shellfish and allow more accurate predictions of depuration times to below permissible limits. When used in conjunction with forecasting models of Pseudo-nitzschia blooms, the predicted levels of domoic acid in shellfish can be better estimated along with the potential economic consequences of recreational and commercial shellfish closures.

     


    Shumway, S.E. (University of Connecticut, Storrs). ECOHAB: Assessment of the potential for introduction of harmful algal bloom (HAB) species via shellfish transport. 1/1/05-12/31/07. EPA Award: RD831704. Email: sshumway@uconnvm.uconn.edu.

    Published records and other data clearly indicate that cells and cysts of many HAB species can pass intact and viable through the digestive tracts of bivalve molluscs. These cells and cysts are capable of establishing new cell cultures under laboratory conditions. Shellfish are routinely transplanted between areas during normal aquaculture, shellfishing, and shellfish restoration practices. While the potential role of bivalves as vectors of HAB species has been recognized by many authors, their actual role in this process has not been examined. We propose a three-year study to assess potential pathways of introduction and consequences of shellfish transfer (commercial or personal) on HAB distribution, i.e. to determine the risk of transferring toxic algal cells/cysts during transport of live bivalves between sites, and to establish and evaluate mechanisms to minimize these risks using best management practices (BMPs). In addition to establishing means of mitigation and control, we have also included a strong outreach education component because we believe that education will play a major role in stemming or slowing the transfer of HAB species by way of shellfish movement.

    Objectives: Our major objectives are to: (1) determine which algal species pass intact and viable through the digestive tract of commercially important bivalve molluscs; (2) determine the extent to which washing and purging shellfish intended for transfer can slow or eradicate the potential transfer of HABs; (3) determine when bivalves are safe to transport following exposure to HAB species; (4) assess field populations in areas of known HAB outbreaks for the presence of viable cells/cysts in resident shellfish populations during non-bloom periods; and (5) provide information to the user groups through presentations, management agencies, trade publications, pamphlets, and web pages.
    Approach: We will address these questions using standard laboratory techniques and well-established experimental protocols to determine rates of uptake, retention, and elimination of toxic cells/cysts, as well as excystment and release from fecal strands and the subsequent culture of viable cells. In collaboration with industry we will design, apply, and evaluate management strategies and BMPs.

    Expected Results: This research will be valuable to aquaculturists, watermen, processors, and managers for public health, and will assist in both habitat management and preservation of habitat integrity. Understanding the vectors for transfer of HAB species is critical to responsible environmental stewardship. This study specifically addresses special emphasis areas 1 and 4(b)prevention and mitigation strategies, and the sources, fates, and consequences of HABs in food webs and fisheries. Results from this study will ensure that the user groups are provided the most current information available, in a useable format, to control and mitigate impacts of HABs on public health, shellfish aquaculture, and the environment.

     


    Thomas, A. and H. Xue (University of Maine). ECOHAB: Oceanographic links to Alexandrium-imposed toxicity in the Gulf of Maine. 9/1/04-8/31/07. NOAA Award: NA04NOS4780271. Email: thomas@maine.edu.

    We propose a data mining project based on remote sensing, numerical modeling and statistical analyses that will identify and quantify links between coastal toxicity caused by Alexandrium in the Gulf of Maine and oceanic variability. The Maine Department of Marine Resources (DMR) monitors toxin levels in multiple species of shellfish at over 300 Maine coastal sites throughout non-winter months. The same sites and/or species are not necessarily sampled each time. Nevertheless, over 25 years of these data provide an unparalleled documentation of HAB variability along the Maine coast and our best window into the interannual variability of Alexandrium dynamics. Annual in situ surveys of potentially relevant oceanographic characteristics to compare with the DMR record are not available. Three sources of data do provide systematic and consistent metrics of oceanographic variability for extensive, overlapping time periods. 1) Satellite data. Our sea surface temperature (SST) image database provides 4-5 images/day, 1985 - today. These data define our study period (1985-2006), allowing analysis of interannual variability in SST and surface thermal patterns indicative of circulation as time/space series within each year. A shorter timeseries (1997-2004) of daily SeaWiFS multispectral data supplement the SST data. 2) Model fields. We will reconstruct major 3D hydrographic structure and circulation in each study year using our Gulf of Maine numerical model. Based on the 3D Princeton Ocean Model, our hindcasts will use meteorological forcing from the NCEP Eta reanalysis, river discharge, assimilation of daily satellite SST fields and climatological open ocean boundary conditions. 3) River discharge and meteorological records in each study year provide coincident ancillary data. The overarching hypothesis that structures our investigation is: Interannual differences in the location, timing and magnitude of toxicity events along the Maine coast are associated with interannual variability in Gulf of Maine oceanographic patterns. We will statistically isolate and quantify dominant patterns in the multidimensional and gappy toxicity record. A suite of metrics indicative of oceanographic structure and forcing will be extracted from the image time series, numerical model output fields and ancillary data (e.g. dominant time/space SST variability, location/strength of specific frontal zones, timing/location of patterns indicative of specific circulation, pycnocline depth, timing of stratification, cross-shelf salinity structure, coherence of alongshore currents, etc.). We then use correlation functions and multivariate statistical tools to identify and quantify those characteristics of Gulf of Maine oceanography most consistently linked to toxicity events. We propose an iterative system of analysis, basing initial approaches on previous ECOHAB results and earlier analyses of subsets of the toxicity record, modifying our metrics as we learn which environmental parameters vary most closely with toxicity timeseries. Our results 1) simplify dominant patterns of variability in the 22+ year toxicity record 2) identify those Gulf of Maine environmental characteristics most closely linked to toxic events 3) deliver a system of easily monitored HAB ocean indices to managers and 4) point to the most promising oceanic features upon which to focus future, more physiologically, based research.

     


     

    Van Alstyne, K.L. (Western Washington University) and T.A. Nelson (Seattle Pacific University). ECOHAB: Harmful ulvoid macroalgal blooms in Washington State: Distribution, environmental effects, and toxin production. 8/1/05-7/31/08. NOAA Award: NA05NOS4781192. Email: kathy.vanalstyne@wwu.edu.

    Blooms of harmful ulvoid green macroalgae occur regularly in Washington (WA) coastal waters and are typically composed of Ulva spp. and Ulvaria obscura. Anecdotal information from aquaculturists suggests they are increasing in magnitude. Their harmful effects towards seagrasses, fish, and invertebrates are generally thought to result from smothering or anoxia. However, recent studies have isolated toxins from bloom-forming algae and found them to be detrimental towards macroalgae, microalgae, and invertebrates. Anecdotal evidence suggests that toxin release by ulvoid blooms may be having community-level impacts.
    Objectives: The goals of our research are to: (1) determine when and where ulvoid blooms occur in the Puget Sound/ Northwest Straits region, (2) determine the physical, chemical, and biological factors associated with blooms, (3) determine the importance of nutrients, light, and herbivores in controlling the growth of ulvoid algae, and (4) examine toxin production by bloom forming algae and the impacts of toxins on co-occurring plants and animals.
    Approach: We will assess spatial and temporal changes in bloom occurrences by analyzing underwater videos taken by the WA Department of Natural Resources from 2000-2008. We will measure environmental factors associated with bloom formation by monitoring sites where blooms typically have and have not occurred in the past. Manipulative field experiments will be used to determine the effects of light and nutrients on the growth and physiology of bloom forming algae. Field measurements will be used to measure grazing rates on algae and bioassay guided fractionation methods will be used to isolate algal toxins. Toxicological assays will be used to assess the effects of toxins on marine plants and invertebrates.
    Expected Results: This research will advance our understanding of the causes, controls, and impacts of green algal blooms in WA, and is likely to be broadly applicable to macroalgal blooms in other locations. Our research will provide a better understanding of spatial variability in harmful macroalgal blooms, of the mechanisms that initiate bloom formation and contribute to their persistence, and of the importance of toxins in mediating the effects of these blooms on surrounding communities.


  • Title:  Role of parasitism on HAB dynamics:  Amoebophrya sp. ex Aexandrium tamarense

    Investigators:  D. Wayne Coasts , Mario R. Sengo and Donald M. Anderson

    Institutions:  Smithsonian Environmental Research Center, Edgewater, MD, Woods Hole Oceanographic Institution, Woods Hole, MA

    Funded:  NOAA NOS NCCOS CSCOR

    Harmful algal blooms (HABs) develop from the rapid growth and accumulation of certain microalgal species, and cause many deleterious impacts on human health, aquatic organisms, important industries, and the quality of freshwater reservoirs and coastal environments.  Globally, HABs have seen a dramatic increase in frequency, magnitude, distribution, and impacts in recent years, which has prompted considerable interest in processes that regulate their formation, persistence, and decline.  Trophic interactions, like parasitism, may play a significant role in bloom dynamics.  Species of Amoebophrya are particularly noteworthy, as they are widely distributed in coastal environments, with infections known for numerous host taxa.  Infections prevent reproduction of the host, are short in duration, and invariably result in death of the host, all of which make these parasites likely candidates for controlling host populations.  These organisms are even viewed as potentially useful agents for the biological control of HABs.

    This proposal focuses on a recently-isolated dinoflagellates parasite from the genux Amoebophrya and its interaction with its host, Alexandrium tamarense, a bloom-forming, toxic dinoflagellates that impacts U.S. coastal waters.  Our first objective is to characterize the parasite in culture to fully establish its identity and relationship to the genus Amoebophrya.  This work includes basic parasite morphology/cytology throughout its entire life cycle, and molecular phylogenetic analysis.  In conjunction with phylogenetic analysis, our second objective is to develop a series of molecular probes, which are Amoebophrya-specific and specific to our iolate, for later us in field studies.  Our third objective is to study host-parasite interactions in the laboratory.  This work examines parasite generation time under various conditions:  host-parasite ratio, growth phase of host, dinospore age, and environmental conditions (i.e., light, temperature, salinity).  The data from this work will be used in modeling the dynamics of host and parasite populations.  Our fourth objective is to examine the host range and preference of our isolate.  Our fifth objective is to determine the fate of saxitoxins from A. tamarense during and after infection.  Lastly, our sixth objective is to study the dynamics of the parasite and A. tamarense (and other dinoflagellate hosts) in the field over time and space to determine the role of parasitism on host populations. 

    Overall, this research seeks to further our fundamental understanding of this little-known group of parasitic organisms, and to investigate the role these parasites play in affecting host population dynamics in the laboratory and field.  These studies, together with practical and ecological considerations, may allow us to evaluate the strategy of using this parasite as a biological agent to control HABs like those produced by A. tamarense.

  • Title:  LyngbyaHAB:  Ecology of toxic marine cyanobacteria Lyngbya spp. in Florida estuarine and coastal waters

    Investigators:  Hans W. Paerl; Valerie J. Paul, John W. Burns; Judith M. O’Neil,

    Institutions:  Univ. of North Carolina at Chapel Hill, Morehead City, NC, PBS&J, Univ. of Maryland Center for Environmental Science, Cambridge, MD

    Funded:  NOAA NOS NCCOS CSCOR

    Benthic cyanobacterial (blue-green algal) HABs are becoming more numerous, widespread and persistent in tropical, subtropical and temperate marine embayments, estuaries, and reef environments. Blooms can have many negative impacts, such as directly overgrowing and smothering seagrass, shellfish habitats, and coral reefs. Some nuisance taxa produce toxins and other bioactive metabolites.  A genus of particular concern is the filamentous, non-heterocystous nitrogen-(N2) fixing cyanobacteria Lyngbya, species of which (e.g. L. majuscula) are distributed worldwide, especially in the tropical and subtropical oceans.  In U.S. coastal waters, Lyngbya blooms have been responsible for fouling large segments of Florida estuaries and bays, the most obvious being Tampa Bay, near-shore reef environments of the Florida Keys, and reefs off of the southeastern coast near Broward County.  There are increasing numbers of reports on finfish and shellfish disease and kills as well as human maladies (skin irritations, intoxication) associated with outbreaks of Lyngbya majuscula blooms and other cyanobacterial bloom species.  Coastal cyanobacterial blooms are thought to be associated with eutrophication and hydrologic modification of freshwater discharge to marine environments. 

        The following objectives will be carried out in our interdisciplinary effort: 1) Nutrient additions in in situ bioassays to determine the nutrient(s) that stimulate Lyngbya and other cyanobacterial blooms; 2) Couple Lyngbya nutrient dynamics with other nearshore HABs (red tides) by identifying the Lyngbya N contribution to these blooms; 3) Isolate and characterize toxins and other bioactive secondary metabolites produced during bloom and non-bloom events; 4) Screen Lyngbya isolates from Florida systems for grazer inhibition, and identify potential pathways for bioaccumulation and biomagnification of toxins; and 5) Sequencing of the N2 fixing gene nifH, to examine potential relationships between specific strains, their N2 fixing activity, toxicity, growth and proliferation within Tampa Bay and other Florida Lyngbya populations.

    Parameters we will investigate have been linked to human perturbations of nearshore environments; hence research products can be evaluated and applied in the context of developing future nutrient and hydrologic management strategies for these habitats and well as risk management plans for humans exposed to Lyngbya toxins.  Research results are applicable to subtropical and tropical ecosystems nationally (Hawaii, American Samoa, Guam, Puerto Rico, U.S. Virgin Islands, Gulf of Mexico and S.E. Atlantic) and internationally (e.g., Australia, South Pacific, Caribbean).

    Title:  Development of PCR and immunofluorescence methods to measure HAB cell concentration and cell division rate:  Karlodinium micrum as a model system

    Investigators:  Senjie Lin and Huan Zhang

    Institution:  University of Connecticut, Groton, CT

    Funded:  NOAA NOS NCCOS CSCOR

    To monitor dynamics of harmful algal blooms (HABs) and to study potential environmental factors regulating the blooms, it is crucial to simultaneously estimate cell concentration and in situ gross growth rate (GGR; i.e., cell division rate) at the early stage of bloom development.  Molecular techniques have proven useful in detecting HAB species and measuring cell concentrations, application of such powerful technology to estimation of GGR is in its infancy.  A Targeted Individual Study is proposed here to develop a PCR and immunofluorescence (IF) detection system that will allow simultaneous quantification of cell concentration and GGR for Karlodinium micrum.  First, extending from the PIs’ previous research, a dual-gene (mitochondrial cytochrome b-ribosomal RNA) PCR primer set will be developed for quantifying cell concentration.  Second, using K. micrum proliferating cell nuclear antigen (PCNA) gene (pcna) recently cloned in the PI’s laboratory, antibodies will be produced to allow immunostaining and estimation of GGR.  Third, protocol for PCNA immunofluorescence will be established; meanwhile, correlation between pcna gene expression (mRNA abundance), measuring Real-Time quantitative RT-PCR, and growth rate will be sought for a second way of GGR estimation.  Fourth, utility of these techniques will be assessed using laboratory cultures and field-collected samples spiked with cultured K. micrum.  The accuracy of the PCR and IF methods for estimating GGR will be evaluated by comparing the estimated with the actual GGR measured from cell counts.  Fifth, taking advantage of archived DNA samples, the dual-gene PCR will be used to study K. micrum spatial and temporal variation in Neuse River, Chesapeake Bay tributaries, and Long Island Sound.  Results from this study will provide specific PCR primers, antibodies, and protocols for studying population dynamics and GGR for K. micrum.  An insight into what environmental factors are conductive to bloom formation of K.  micrum is likely to be achieved.  The results will also provide a framework based on which similar protocols can be established for other HAB species.  Two gratudate students will be trained on monitoring HAB cell concentration and GGR using molecular techniques (one supported this project, another supported by the Department as a TA)  Novel information gained from this study will be incorporated to courses taught by the PI.Title:  Harmful Ulvoid Macroalgal Blooms in Washington State:  Distribution, Environmental Effects, and Toxin Production


  • Investigators:  Kathryn L. Van Alstyne and Timothy A. Nelson

    Institutions:  Western Washington University Bellingham, WA, Seattle Pacific University, Seattle WA

    Funded:  NOAA NOS NCCOS CSCOR

    Blooms of harmful ulvoid green macroalgae occur regularly in Washington (WA) coastal waters and are typically composed of Enteromorpha spp., Ulva fenestrata, and Ulvaria obscura.  Anecdotal information from aquaculturists suggests they are increasing in magnitude.  Their harmful effects towards seagrasses, fish, and invertebrates are generally thought to result from smothering or anoxia.  However, recent studies have isolated toxins from bloom-forming algae and found them to be detrimental towards macroalgae, microalgae, and invertebrates.  Anecdotal evidence suggests that toxin release by ulvoid blooms may be having community-level impacts. 

    Objectives:  The goals of our research are to: (1) determine when and where ulvoid blooms occur in the Puget Sound/ Northwest Straits region, (2) determine the physical, chemical, and biological factors associated with blooms, (3) determine the importance of nutrients, light, and herbivores in controlling the growth of ulvoid algae, and (4) examine toxin production by bloom forming algae and the impacts of toxins on co-occurring algae, invertebrates and seagrasses. 

    Approach:  We will assess spatial and temporal changes in bloom occurrences by analyzing underwater videos taken by the WA Department of Natural Resources from 2000-2007.  We will measure environmental factors associated with bloom formation by monitoring sites where blooms typically have and have not occurred in the past.  Manipulative field experiments will be used to determine the effects of light and nutrients on the growth and physiology of bloom forming algae.  Field measurements will be used to measure grazing rates on algae and bioassay guided fractionation methods will be used to isolate algal toxins.  Toxicological assays will be used to assess the effects of toxins on macroalgae, seagrasses, invertebrates, and fish. 

     ExpectedResults:  This research will advance our understanding of the causes, controls, and impacts of green algal blooms in WA, and is likely to be broadly applicable to macroalgal blooms in other locations.  Our research will provide a better understanding of spatial variability in harmful macroalgal blooms, of the mechanisms that initiate bloom formation and contribute to their persistence, and of the importance of toxins in mediating the effects of these blooms on surrounding communities. 





  • Last updated: May 15, 2013